Cytoplasmic regulatory factors appear to play an important role in the early development of fertilized eggs. We feel they also have an important role in differentiation and our studies attempt to elucidate this role using cultured mammalian cells as a model system. We have used cybrids for study of the control of differentiated functions by cytoplasmic regulatory factors. Extinction of hemoglobin induction and activation of phenylalanine hydroxylase (PH) in mouse erthroleukemia (MEL) cybrids suggest the existence of self perpetuating negative and positive cytoplasmic regulatory factors respectively. MEL cells, selected in tyrosine free medium after fusion with rat hepatoma cytoplasm, express phenylalanine hydroxylase constitutively on continuous culture. Isoelectric focusing and immunotitration studies indicate that the cybrids express the mouse form of the enzyme. Peptide map analysis of PH from rat hepatoma and MEL cybrids will be carried out to establish unequivocally that the enzyme expressed in MEL cybrids is due to true activation of the normally silent PH gene in MEL cells. Using physical and chemical agents to inactivate proteins and nucleic acids, we are presently attempting to identify the chemical nature of the cytoplasmic regulatory factor for PH gene. Our preliminary findings indicate that the cytoplasmic regulatory factor for PH is perhaps RNA in nature. We hope to exploit cybrid technology to the maximum to gain a better understanding of how cellular functions are regulated.